Title: Controllable sequential small molecule and RNA therapeutics Project Summary: Nanoparticles are well-established delivery vehicles capable of shuttling molecular cargoes across different types of biological membranes. A major flaw of the standard nanoparticle assembly is a lack of selective control over cargo release. Functional cargoes, such as drug molecules, are typically unloaded and activated in response to endogenous factors, including pH, redox potential and ATP gradient. However, biological variability between different types of cells and tissues causes uncertainty with regards to the timing, location, degree and rate of payload release. Lack of control over this process can result in premature payload activation before the nanoparticles have reached their intended target. In turn, this can lead to a highly undesirable toxicity, loss of valuable drug payload in the body and a necessity to increase the drug dosage. In addition, incomplete drug release, in response to dynamic endogenous factors, leads to inefficient therapeutics and also necessitates an increase in the dose administration. The ultimate goal of this proposal is to address these flaws using a transformative approach involving bio-orthogonal chemistry. The two-step reaction readily proceeds in a physiological environment on a minute timescale and is completely inert to functional groups. This reaction can be precisely controlled and chemical design of the bio-orthogonal groups provides flexibility of the release (activation) kinetics; slow or fast. The approach has the potential to revolutionize the currently used in vivo drug activation approaches and nanoparticle-based delivery methods. We are proposing to use this chemical approach to activate three types of pro-drugs: siRNA against EGFR, LNA-based anti-sense therapeutic oligonucleotides (ASO) against miR-10b, erlotinib as a small molecule inhibitor against EGFR and doxorubicin as a small molecule chemotherapeutic agent. Superparamagnetic iron oxide nanoparticle (MNPs), derived from clinically used MRI contrast agents, will be used as the nanocarriers. Small molecule non-toxic tetrazine will be employed as an exogenous chemical trigger. In vitro studies will be carried out using triple negative breast cancer as one of the most challenging systems to treat. Combinations of the aforementioned drugs will be evaluated for their synergistic properties. The drugs will be activated simultaneously or sequentially using a single dose of tetrazine to observe the greatest therapeutic response. The image-guided technology described in this proposal is transformative in the following aspects of nanomaterial-based payload delivery: 1. Our methodology is built on a biocompatible and highly specific chemical reaction, which can trigger drug activation, regardless of the depth of the target tissue. The rate, slow or fast, of the drug activation can be controlled using a single chemical trigger. This will enable one to control the activation of two drugs, simultaneously or sequentially, with subsequent administration of a single chemical trigger. Furthermore, the bio-orthogonal inverse electron demand Diels-Alder reaction will allow for complete payload release. 2. The release will be triggered only when the temporal delivery of the nanodrugs is validated. Additionally, our approach has the ability to monitor the in vitro drug activation using fluorescence microscopy. Such information will enable one to understand the timing and rate of drug release and revisit the drug design and administration schedule. Current approaches aim to address only imaging of drug delivery whereas we aim to monitor both the drug delivery and its activation. In summary, our strategy is superior to the existing approaches that rely on an uncontrolled payload release and potentially premature activation of drugs in living systems. This proposal will focus on the development of a general paradigm. However once fully optimized, the engineered technology could be translated to different diseases to explore the delivery of a wide range of drugs. In addition, the bio-orthogonally releasable linker could be installed into a variety of different nanomaterial platforms, such as quantum dots or polymeric nanoparticles. Different imaging modalities could be utilized to visualize payload delivery.